Generation of humanized liver mouse model by transplant of patient-derived fresh human hepatocytes.

Some research groups have produced immunodeficient mice with human liver tissue as a model system for the analysis of drug metabolism and liver regeneration. Mouse models are important for research and development of drugs and vaccines for viral infections. Recent progress in developing humanized mouse models permits studies of adaptive immune responses, innate host responses, and therapeutic approaches for several liver diseases of viral etiology. In this study, we generated a humanized liver mouse model by transplant with fresh patient-derived hepatocytes (1 × 10(6) cells/mouse, intrasplenic injection) into preconditioned (50 mg/kg ganciclovir, intraperitoneal injection) mice (herpes simplex virus type 1 thymidine kinase [TK] transgene expressed within the liver of a highly immunodeficient mouse strain [NOG]). Successful reconstitution of human hepatocytes in TK-NOG mouse liver tissues was observed with a strong proliferation of human cells in a time-dependent manner, using cytokeratin 8/18 stain. Similarly, we detected significantly increased human albumin levels in TK-NOG mouse liver tissue and blood sera on immune staining and enzyme-linked immunosorbent assay. Therefore, this humanized liver mouse model provides a biomedical tool for studying human liver physiology, drug metabolism, and liver pathogenesis of viral etiology or liver regeneration.

HLA-A*33:25, a novel allele identified by sequence-based typing, is a well-documented allele in Koreans.

The new allele, HLA-A*33:25, differs from A*33:03:01 by one nucleotide substitution in exon 4 (c.778G>A) which results in an amino acid change at codon 236 [GCA→ACA (Ala>Thr)] in ß2-microglobulin-binding domain.

Monitoring of cytomegalovirus-specific CD8+ T-cell response with major histocompatibility complex pentamers in kidney transplant recipients.

INTRODUCTION: Cytomegalovirus (CMV) can reactivate causing serious clinical problems during immunosuppression. CMV-specific CD8(+) T cells play an important role in the control of CMV reactivation. Using pentameric major histocompatibility complex (MHC) peptide complexes, we investigated cellular immune responses to CMV among healthy individuals and kidney transplantation recipients in Korea, which is an endemic area of CMV infection. MATERIALS AND METHODS: Analysis of CMV-specific T cells was performed on 28 healthy individuals and 40 recipients who bore human leukocyte antigen (HLA)-A2 or -A24. CMV pp65 pentamer-binding cells incubated with various monoclonal antibodies were measured by four-color flow cytometry. RESULTS: Detectable levels of pentamer(+) CD8(+) T cells were present in 109/139 samples (78.4%) that stained with the A*02NLV-pentamer, while 15/67 samples (22.4%) stained with the A*24QYD-pentamer (P < .01). Among patients with HLA-A2, 22/24 (91.7%) samples showing positive CMV antigenemia revealed detectable pentamer(+) CD8(+) T cells, while 87/115 (75.7%) displaying negative CMV antigenemia had detectable pentamer(+) CD8(+) T cells (P = .04). There was no significant difference in percentages of pentamer(+) CD8(+) T cells between patients who did versus who did not experience episodes of CMV infection. The subpopulation of CMV-specific CD8(+) T cells in transplantation recipients was evaluated using phenotypic markers; memory cells comprised the majority of the CMV-specific CD8(+) T-cell population. CONCLUSION: The A*02NLV-pentamer complex was useful to monitor CMV-specific T cells. However, MHC pentamer-based techniques did not provide a clear distinction between patients who are or are not at risk for CMV infection.

Development of functional human immune system with the transplantations of human fetal liver/thymus tissues and expanded hematopoietic stem cells in RAG2-/-gamma(c)-/- MICE.

BACKGROUND: There is an increasing need for suitable animal models for the study of the human immune system and disease. The purpose of this study was to develop a practical in vivo model of human immune cell repopulation using ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells and subrenally coimplanted fetal liver/thymus tissues. METHODS: Freshly isolated fetal liver-derived CD34(+) hematopoietic stem cells were frozen until injected and ex vivo expanded with various cytokines for 7 days. After fetal liver/thymus tissues were subrenally coimplanted into preirradiated Rag2(-/-)gamma(c)(-/-) mice, frozen and ex vivo expanded CD34(+) cells were injected intravenously. The peripheral blood of the mice was monitored for the detection of human cell engraftment using flow cytometry. Then we confirmed human T-cell function by in vitro function assays. RESULTS: After fetal liver/thymus tissues were coimplanted into the irradiated Rag2(-/-)gamma(c)(-/-) mice, with frozen and ex vivo expanded CD34(+) hematopoietic stem cells, human cell engraftments were determined using hCD45 and multilineage markers. The cultured cells with the cytokine combination of stem cell factor, thrombopoietin, Flk2/Flk3 ligand (FL), and interleukin-3 showed stable and long-term engraftment compared to other combinations. The ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells, under our culture conditions, accomplished a large volume of expanded cells that were sustained, demonstrating self-renewal of the evaluated markers, which may have indicated long- term repopulation activity. CONCLUSION: The results of this study demonstrated a practical mouse model of expanded human immune cells especially T cells in Rag2(-/-)gamma(c)(-/-) mice.

Reconstitution of human lymphocytes following ex vivo expansion of human umbilical cord blood CD34+ cells and transplantation in Rag2-/- gamma c-/- mice model.

BACKGROUND: Due to ethical issues, in vivo studies of the human immune system have been difficult. Thus, small-animal xenotransplantation models have been employed, although they scarcely sustain a human immune response. In this study, we compared human cell repopulation tendencies and functionality in Rag2-/- gamma c-/- mice following various ex vivo expanded human hematopoietic stem cells (HSCs). METHODS: Human umbilical cord blood (UCB) CD34+ cells were cultured for 7 days with a cytokine combination of stem cell factor, Flk2/Flt3 ligand, and thrombopoietin, with absence or presence of rhIL-3, then transplanted into Rag2-/- gamma c-/- mice. Reconstituted human lymphocytes were analyzed based on the expression of CD45 as well as CD3, CD19, and CD56 in peripheral blood (PB) until 16 weeks after transplantation. BrdU assay and functional analysis of reconstituted human lymphocytes used PHA- or rhIL-2-stimulated splenocytes and bone marrow cells from recipient mice. RESULTS: The percentage of human CD45dim cells, not CD45bright cells, in PB of mice transplanted with cultured HSCs with rhIL-3 was much higher than in the group without rhIL-3 (approximately 2.5-fold at week 10 posttransplantation). The humanized mice showed systemic repopulation with a comprehensive array of human lymphohematopoietic cells, including T, B, natural killer (NK) cells, and even dendritic cells. However, the expression level was also dim. The number of CD3+ T cells and CD56+ NK cells was especially increased in the presence of rhIL-3. In addition, after in vitro restimulation proliferation assays and NK activity of interferon-gamma secretion showed greater effects in the presence of rhIL-3. CONCLUSION: These data suggested that the development of a diverse repopulation of human lymphocytes was possible in Rag2-/- gamma c-/- mice after transplantation of cultured UCB CD34+ HSCs with interleukin-3.

The 'four-in-one' procedure for habitual dislocation of the patella in children: early results in patients with severe generalised ligamentous laxity and aplasis of the trochlear groove.

We describe our experience with the 'four-in-one' procedure for habitual dislocation of the patella in five children (six knees). All the patients presented with severe generalised ligamentous laxity and aplasia of the trochlear groove. All had a lateral release, proximal 'tube' realignment of the patella, semitendinosus tenodesis and transfer of the patellar tendon. The mean age at the time of the operation was 6.1 years (4.9 to 6.9), and the patients were followed up for a mean of 54.5 months (31 to 66). The clinical results were evaluated using the Kujala score. There has been no recurrence of dislocation. All the patients have returned to full activities and the parents and children were satisfied with the clinical results. The mean Kujala score was 95.3 (88 to 98). Two patients had marginal skin necrosis which healed after debridement and secondary closure. These early results in this small group have shown that the 'four-in-one' procedure is effective in the treatment of obligatory dislocation of the patella in children with severe ligamentous laxity and trochlear aplasia.

Infarct patterns in atherosclerotic middle cerebral artery versus internal carotid artery disease.

OBJECTIVE: To compare clinical and radiologic characteristics of atherosclerotic middle cerebral artery (MCA) vs internal carotid artery (ICA) disease. METHODS: The authors defined atherosclerotic MCA and ICA disease as >50% symptomatic stenosis or occlusion without significant ICA and MCA stenosis on MR angiography. Patients with potential cardiac sources of embolism were excluded. The authors analyzed clinical, laboratory, and neuroradiologic data of the two groups. RESULTS: Among the 920 consecutive patients with acute ischemic strokes, 112 met the criteria for atherosclerotic MCA and 71 met the criteria for ICA disease. Clinically, the MCA group more frequently presented with lacunar syndrome (p = 0.001), whereas the ICA group more often presented with total anterior circulation infarct and had higher initial NIH Stroke Scale scores than the MCA group (all p < 0.001). Whereas deep perforator and internal border-zone infarcts were associated with MCA disease (p < 0.001 and 0.012), territorial infarcts and superficial perforator infarcts were associated with ICA disease (p < 0.001 and p = 0.009). The topographic patterns with respect to the degree of stenosis were also significantly different between the two groups. CONCLUSIONS: The clinical and radiologic stroke patterns were distinctively different between atherosclerotic MCA and ICA disease, suggesting different underlying pathogeneses.